Executive Summary : | Vitamin B2 (riboflavin) is an essential micronutrient required by living organisms to catalyze electron transfer reactions in energy metabolism and in oxidation-reduction reactions in the cell. The active cofactor forms of vitamin B2 which are bound and utilized by enzymes in our cells are flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). The structure of FAD consists of an isoalloxaxine ring, which is responsible for the cofactor’s oxidation-reduction properties, and a ribityl chain connected with an adenosine moiety via two phosphates. FAD biosynthesis consists of two steps – i. the first step is the conversion of riboflavin to FMN via a phosphorylation reaction typically with adenosine triphosphate (ATP) as a co-substrate and ii. the second step is where FMN is further adenylated by a second molecule of ATP to produce FAD. Both these steps can either be catalyzed by individual monofunctional enzymes as found in humans or by a single bifunctional enzymes as found in many bacteria. However, very little is known about the reason for the choice of ATP as the phosphorylating and adenylating agent as compared to other cellular nucleotide triphosphates (NTPs) such as guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP). What if GTP or CTP were used in the second step, consequently resulting in flavin analogs such as flavin guanine dinucleotide (FGD) or flavin cytidine dinucleotide (FCD) under cellular environments? If we provided a fluorescently labeled unnatural nucleotide, can we synthesize a fluorescent flavin dinucleotide in the cell? Can we alter the nucleotide specificity of the FAD biosynthesis enzymes to make alternate unnatural FAD analogs?
NTPs such as ATP, GTP, CTP, and UTP are commonly encountered in biochemistry not only the building blocks of RNA, but also function as energy providers for cellular processes and mediators in important signaling pathways. The energy from the transfer or hydrolysis of the phosphate group of the NTP is enzymatically coupled with unfavorable biochemical reactions to facilitate their progress in the cell. In such reactions, even though the nucleobase (adenine, guanine etc.) does not appear to play a direct role, the enzyme exhibits high selectivity for a specific NTP. We hypothesize that the choice of NTP could be governed by the active site residues that bind to the nucleobase or by the cellular concentration of the NTPs. In this proposal, we wish to interrogate the molecular basis of the choice of nucleotide by an enzyme using a mechanistic approach. Specifically, we wish to identify the residues responsible for NTP specificity in the microbial FAD biosynthesis pathway enzymes. Using protein engineering, we want to alter these enzymes to make unnatural analogs of FAD, which are candidate molecules for the development of antimicrobials drugs and for synthetic biology applications. |